We have prepared compounds which inhibit aromatase (estrogen synthetase) in vitro and have found them to be highly effective in causing regression of DMBA-induced, hormone dependent mammary tumors of the rat. Evidence suggests that aromatase inhibitors act in vivo by inhibiting estrogen biosynthesis: measurements of ovarian secretion were significantly reduced by inhibitor treatment compared to controls and peripheral gonadotropin levels are similar in treated and control rats. Also, when exogenous estradiol was given in addition to inhibitor treatment, tumor regression was prevented. In order to develop aromatase inhibitors as useful compounds for breast cancer therapy and other diseases influenced by estrogen, we propose to investigate further the mechanism of action of aromatase inhibitors. We will determine whether the compounds can inhibit estrogen biosynthesis in vivo by their direct infusion into the externalized ovary of the sheep. We will also determine whether peripheral aromatization can be inhibited in the primate with our present inhibitors. We propose to investigate whether any of the properties of the inhibitors in vivo can be attributed to androgenic activities or whether they can compete for steroid hormone receptors. Using radio-labelled aromatase inhibitors, we are studying the extent of metabolism, distribution and clearance rate of the compounds and whether metabolites contribute to the activity of the inhibitors. By utilizing our structure-activity data we expect to synthesize more potent compounds. An important aspect of our program is the use of affinity labelling and other methods to prepare irreversible inhibitors. We will study the effects of combining aromatase inhibitors with other anticancer agents to determine if more complete and permanent regression can be obtained. We also propose to apply aromatase inhibitors to studying the interrelationships of estrogen with other hormones and their effects on steroid hormone plasma and receptor levels.